Microorganism, lactamase enzyme obtained therefrom, and their use

ABSTRACT

A lactamase enzyme having good stability, capable of hydrolysing an enatiomer of the bicyclic lactam, 2-azabicyclo[2.2.1]hept-5-en-3-one, to give (-) lactam and (+) amino acid, has been found in a strain of Comamonas acidivorans. The enzyme has been isolated and cloned, and its structure identified.

FIELD OF THE INVENTION

This invention relates to a microorganism, lactamas enzyme obtainedtherefrom, and their use.

BACKGROUND OF THE INVENTION

The bicyclic γ-lactam, 2-azabicyclo[2.2.1]hept-5-en-3-one, is a usefulsynthon that can be used for the production of carbocyclic nucleosideswhich are gaining in importance as therapeutic agents. Published areasto which such nucleosides are being targeted include antivirals (e.g.Vince and Hua, J. Med. Chem., 33:17-21 (1990), against e.g. HIV) andcardiac vasodilators (adenosine agonists). A major benefit of thecarbocyclic ring in such agents is its resistance to breakdown byenzymes in the body. By comparison, naturally-occurring ribosylnucleosides may be more readily cleaved by nucleases, so that theirbioactivity is lost.

Although carbocyclic nucleosides are known in nature, e.g. Aristeromycinfrom Streptomyces citricolor, natural yields tend to be low and theisolated products have then to be further manipulated to obtain moreuseful compounds. A more economic route is to synthesise the requiredcompounds chemically, starting from the γ-lactam. However, as chemicallysynthesised, γ-lactam is racemic. By conventional synthesis, theultimate drug will also be a mixture of enantiomers, which causesregulatory concerns if one of the enantiomers is not very active orcauses unwanted side-effects. There is a need therefore to put a stepinto the synthesis where either of the two enantiomers of a racemicsynthon can be isolated and the rest of the drug then built on it.

An effective way of doing this is to use an enzyme to selectivelyhydrolyse one enantiomer of the racemic γ-lactam across the amide bond,to give the cyclic amino acid compound and leave the other enantiomer.The remaining lactam can then be readily separated from the amino acidproduct by extraction into dichloromethane, purified by crystallisationand used in subsequent downstream chemistry to build up the requireddrug. By careful selection of the right enzyme it is possible to find anenzyme highly selectively for only one of the lactam enantiomers suchthat at marginally greater than 50% conversion, lactam of high ee (>90%)remains. Enzymes have been found that are selective for either of thetwo enantiomers.

EP-A-0424064 discloses methods for carrying out the above describedresolution and provides two organisms that produce enzymes that have thedifferent selectivities. A Rhodococcus strain produces an enzyme whichhydrolyses the (-) lactam, enabling the (+) lactam to be isolated forfurther use, whereas a Pseudomonad produces an enzyme which hydrolysesthe (+) lactam, enabling isolation of the (-) lactam.

Further enzymes that carry out these active hydrolyses have also beendescribed in the literature. Thus Taylor et al, Tetrahedron; Asymmetry,4(6):1117-1128 (1993), describe an enzyme selective for hydrolysis ofthe (+) lactam from Pseudomonas fluorescens and an enzyme selective forthe (-) lactam from a strain of Aureobacterium. A further enzymeselective for the hydrolysis of the (+) lactam has been described byBrabban et al. J. Ind. Microbiology. 16:8-14 (1996).

In order to develop a robust industrial biotransformation process, it isdesirable to use an enzyme or whole cell biocatalyst that is relativelystable. This can enable biocatalyst recycling and re-use throughimmobilisation, thus greatly reducing biocatalyst cost and enablinghandling of the biocatalyst on a large scale without significant lossesof activity. It is also often found that more stable biocatalysts arebetter able to tolerate high substrate and/or product concentrationswithout inactivation. This then enables biotransformations to be run atthe highest concentration of reactants possible, given kinetic andhandling constraints. This has two advantages: it results in minimalreactor volume requirements and also minimises liquid handing volumesduring product work-up.

Taylor et al, supra, describe a lactamase from Aureobacterium speciesthat is very stable at elevated temperatures and which selectivelyhydrolyses the (-) γ-lactam, giving the (+) γ-lactam and (-) amino acidas a product. The enzyme from this organism has been immobilised andmaintains its stability over months of operation. No enzyme with goodstability and the opposite selectivity is known, although Brabban et al,supra, screened a number of different potential isolates. Previous workwith Pseudomonad type organisms displaying the required lactamaseactivity had shown them to have poor stability. This is unfortunatesince it is the (-) γ-lactam which is the more useful synthon, havingthe more natural stereochemistry and making it easier to build upfunctionality than for instance the (-) amino acid formed by the actionof the Aureobacterium lactamase. There is therefore a need for a stableγ-lactamase with high selectivity for the hydrolysis of the (+) bicyclicγ-lactam.

SUMMARY OF THE INVENTION

Surprisingly, it has been found that a strain of Comamonas acidovorans,which was isolated from the environment, produces an enzyme of highpotential for use in an industrial process for resolution of therequired γ-lactam. This enzyme is not only much more temperature-stablethan previously identified (+) γ-lactamases, but it also enables thebioresolution to be carried out at very high substrate/productconcentrations. This organism has been deposited at the NCIMB, 23 St.Machar Street, Aberdeen, UK, on Aug. 30, 1996, under the terms of theBudapest Treaty, where it has been given the accession number NCIMB40827.

The gene encoding the γ-lactamase has been isolated and sequenced (seeSEQ ID NO:1), and the enzyme's amino-acid sequence derived (see SEQ IDNO:2). This invention relates to compounds having this structure, andfragments thereof having the same activity, as will be readily evidentto one of ordinary skill in the art. The novel enzyme is characterisedby its stability, i.e. one or more of the following:

greater than 85% retention of activity after being held at 40° C. for 4hours or greater than 30% activity after being held at 60° C. for 4hours;

hydrolysis at an initial concentration of 100 g racemic lactam plus 300ml buffer and continuing to at least 90% hydrolysis of the (+) lactamwith less than 5% hydrolysis of the (-) lactam.

DESCRIPTION OF THE INVENTION

The novel enzyme is useful for the enantiospecific hydrolysis of amixture of enantiomers of the required γ-lactam, e.g. a racemic mixture.After reaction, the residual (-) lactam may readily be separated fromthe (+) amino-acid formed by hydrolysis. Both these reactions may beconducted under conditions known to those of ordinary skill in the art.

The enzyme may be used in whole cell or isolated form. It may beimmobilised, if desired, by methods known to those of ordinary skill inthe art.

The enzyme may be produced from the deposited organism. Alternatively,it may be produced by recombinant techology.

Using the DNA and amino-acid sequence provided herein, a person skilledin the art can readily construct fragments or mutations of the genes andenzymes disclosed herein. These fragments and mutations, which retainthe activity of the exemplified enzyme, are within the scope of thepresent invention. Also, because of the redundancy of the genetic code,a variety of different DNA sequences can encode the amino-acid sequencesdisclosed herein. It is well within the skill of one of ordinary skillin the art to create these alternative DNA sequences encoding the same,or similar, enzymes. These DNA sequences are within the scope of thepresent invention. As used herein, reference to "essentially the same"sequence refers to sequences which have amino-acid substitutions,deletions, additions or insertions which do not materially affectactivity. Fragments retaining activity are also included in thisdefinition.

The genes of this invention can be isolated by known procedures and canbe introduced into a wide variety of microbial hosts. Expression of thegene results, directly or indirectly, in the intracellular productionand maintenance of the enzyme. The gene may be introduced via a suitablevector into a microbial host.

A wide variety of ways are available for introducing the gene into themicroorganism host under conditions which allow for stable maintenanceand expression of the gene. A DNA construct may include thetranscriptional and translational regulatory signals for expression ofthe gene, the gene under their regulatory control and a DNA sequencehomologous with a sequence in the host organism, whereby integrationwill occur, and/or a replication system which is functional in the host,whereby integration or stable maintenance will occur.

In the direction of transcription, namely in the 5' to 3' direction ofthe coding or sense sequence, the construct can involve thetranscriptional regulatory region, if any, and the promoter, where theregulatory region may be either 5' or 3' of the promoter, the ribosomalbinding site, the initiation codon, the structural gene having an openreading frame in phase with the initiation codon, the stop codon(s), thepolyadenylation signal sequence, if any, and the terminator region. Thissequence as a double strand may be used by itself for transformation ofa microorganism host, but will usually be included with a DNA sequenceinvolving a marker.

The gene can be introduced between the transcriptional/translationalinitiation and termination regions, so as to be under the regulatorycontrol of the initiation region. This construct can be included in aplasmid, which could include at least one replication system, but mayinclude more than one, where one replication system is employed forcloning during the development of the plasmid and the second replicationsystem is necessary for functioning in the ultimate host. In addition,one or more markers may be present, as described above. Whereintegration is desired, the plasmid will desirably include a sequencehomologous with the host genome.

The transformants can be isolated in accordance with conventional ways,usually employing a selection technique, which allows for selection ofthe desired organism as against unmodified organisms or transferringorganisms, when present. The transformants then can be tested foractivity.

Suitable host cells include prokaryotes and eukarotes. An example is E.coli.

The following Examples illustrate the invention.

1. Isolation of Potetial γ-Lactamase Producing Strains

Approximately 1 g of soil from a ditch was mixed with 20 ml 50 mMpotassium phosphate buffer, pH7, mixed well and shaken at roomtemperature for 30 minutes. A 0.4% inoculum of this suspension was thenplaced into 25 ml enrichment medium in a conical flask and shaken at 30°C. for 41 hours. The following enrichment medium was used:

    ______________________________________                                                         (g.l.sup.-1)                                                 ______________________________________                                        Yeast extract      0.1                                                        NH.sub.4 Cl        2.0                                                        KH.sub.2 PO.sub.4  7.0                                                        Na.sub.2 HPO.sub.4 2.0                                                        MgSO.sub.4         0.4                                                        CaCl.sub.2         0.2                                                        Trace element solution                                                                           0.2                                                        Racemic bicyclic γ-lactam                                                                  2.0                                                        5M NaOH            to pH 7                                                    ______________________________________                                    

The trace element solution comprised:

    ______________________________________                                                      (g.l.sup.-1)                                                    ______________________________________                                               CaCl.sub.2.2H.sub.2 O                                                                  3.6                                                                  ZnO      2.0                                                                  CuCl.2H.sub.2 O                                                                        0.85                                                                 Na.sub.2 MoO.2H.sub.2 O                                                                4.8                                                                  MnCl.sub.2.4H.sub.2 O                                                                  2.0                                                                  FeCl.sub.3.6H.sub.2 O                                                                  5.4                                                                  H.sub.3 BO.sub.3                                                                       0.3                                                                  CoCl.sub.2.6H.sub.2 O                                                                  2.4                                                                  Conc HCl 250 ml                                                        ______________________________________                                    

A 0.5% inoculum was then transfered into a second enrichment flask (25ml) of the same medium, and grown for a further 94 hours. At this point,samples were taken from the flask, diluted in 10 mM phosphate buffer, pH7.0 and plated onto the following medium:

    ______________________________________                                                        (g.l.sup.-1)                                                  ______________________________________                                        Yeast extract     0.1                                                         NH.sub.4 Cl       2.0                                                         KH.sub.2 PO.sub.4 7.0                                                         Na.sub.2 HPO.sub.4                                                                              2.0                                                         MgSO.sub.4        0.4                                                         CaCl.sub.2        0.2                                                         Trace element solution                                                                          0.2                                                         Noble Agar        15.0                                                        5M NaOH           to pH 7                                                     ______________________________________                                    

2.0 g.l⁻¹ N-acetyl-L-phenylanaline was then filter sterilised into theabove autoclave medium on cooling, prior to pouring the plates. After 6days incubation at 30° C., colonies were picked, and purified on furtheragar plates and then used in the screening study.

2. Screening of Recovered Isolates

Isolated colonies were grown in the following medium:

    ______________________________________                                                         (g.l.sup.-1)                                                 ______________________________________                                        Yeast extract      5.0                                                        NH.sub.4 Cl        2.0                                                        KH.sub.2 PO.sub.4  7.0                                                        Na.sub.2 HPO.sub.4 2.0                                                        MgSO.sub.4         0.4                                                        CaCl.sub.2         0.2                                                        Trace element solution                                                                           1.0                                                        Racemic bicyclic γ-lactam                                                                  2.0                                                        Glucose            10.0                                                       5M NaOH            to pH 7                                                    ______________________________________                                    

A colony was inoculated into 4 ml filter-sterilised medium in a sterileplastic container and grown for about 24 hours in a shaker at 30° C.

Cultures were then centrifuged and the pellet resuspended in 1 ml 50 mMphosphate buffer, pH7. To this was then added 1 ml 100 g.l⁻¹ racemicbicyclic γ-lactam in a similar buffer. Reactions were carried out at 30°C. with shaking. Samples were taken over the next 7 days and assayed forconversion of the lactam by HPLC. For those reactions showingsignificant hydrolysis, enantiomeric excess (ee) was determined by GC.

One strain which was isolated showed desirable characteristics. In theinitial screen this strain achieved 52% conversion of the addedsubstrate after 144 hours biotransformation, and the residual lactam wasshown to be the (-) enantiomer with an ee of >99%. Identification by theNCIMB showed the organism to be a strain of Comamonas acidovorans. Thisstrain has been deposited at the NCIMB, as described above.

The following analytical methods were employed:

Extent of Hydrolysis (HPLC). Samples were diluted as appropriate and 20μl injected onto a 15 cm Kromasil C-8 column. The elution buffer was 50%methanol in 10 mM phosphate buffer, pH 7; flow rate 1 ml.min⁻¹ ; runtime 5 minutes. Detection was at λ=225 nm.

ee of reaction products (GC). Samples were extracted into ethyl acetate,dried with anhydrous magnesium sulphate and injected onto a 50 m CPCyclodextrin capillary column. The oven temperature was increased froman initial 140 to 200° C. during the analysis.

3. Fermentation

Seed flasks were prepared using the following medium:

    ______________________________________                                                      (g.l.sup.-1)                                                    ______________________________________                                               Yeast extract                                                                          10                                                                   (NH.sub.4).sub.2 SO.sub.4                                                              1                                                                    KH.sub.2 PO.sub.4                                                                      5                                                                    MgSO.sub.4.7H.sub.2 O                                                                  0.1                                                                  CaCl.sub.2.2H.sub.2 O                                                                  0.05                                                                 Trace elements                                                                         0.1                                                                  NaOH     to pH 7                                                       ______________________________________                                    

The trace element solution is as defined above, except that the amountof conc. HCl is 333 ml.l⁻¹.

75 ml medium was prepared in a 500 ml conical flask. Flasks wereinoculated with the organism, and incubated with shaking at 25° C. tillan absorbance (520 nm) of between 3.5 and 7 had been achieved. Cellswere then inoculated at 0.1% into the fermenter having 1.5 L of thefollowing (sterilised) medium:

    ______________________________________                                                      (g.l.sup.-1)                                                    ______________________________________                                               Yeast Extract                                                                          20                                                                   (NH.sub.4).sub.2 SO.sub.4                                                              2                                                                    KH.sub.2 PO.sub.4                                                                      5                                                                    MgSO.sub.4.7H.sub.2                                                                    0.5                                                                  CaCl.sub.2.2H.sub.2 O                                                                  0.1                                                                  Trace elements                                                                         1.0                                                                  Succinic acid                                                                          10                                                                   PPG 2025 2 ml                                                                 NaOH     to pH 7                                                       ______________________________________                                    

Initial temperature was 25° C. and the pH was controlled at 7.1. Aconstant air flow rate of about 0.5 vvm was maintained, with theagitation being varied between 500 and 1000 rpm to maintain aerobicconditions. After 18.6 hours, a slow feed of concentrated yeast extractwas initiated at a rate equivalent to 2 g yeast extract added perinitial litre per hour, i.e. 3 g per hour. The fermentation wascompleted 24 hours later, the cells harvested by centrifugation andstored as a cell paste in the freezer for further use. A total biomassof about 82 g wet cells was collected and the final fermentationactivity yield was 0.45 U.ml⁻¹ (where 1 U is 1 μmole γ-lactam hydrolysedper minute).

4. Temperature Stability

35.8 g of cell paste was thawed and added to 700 ml lysis buffer,containing 10 mM sodium phosphate (pH 7), 10 mM EDTA, 0.1% Triton X-100,5 mM dithiothreitol and 1 mg.ml⁻¹ lysozyme. The lysis buffer was stirredat room temperature for 5.5 hours, then 37 ml of a 5% solution ofpolyethylenimine, adjusted to pH 7 with HCl, was added and stirred for afurther hour before recovering the supernatant by centrifugation.

To 500 ml supernatant was slowly added 174 g ammonium sulphate with goodmixing to dissolve the salt. After 20 minutes, the precipitate washarvested by centrifugation and resuspended with 100 ml 10 mM sodiumphosphate, pH 7. This was then dialysed against 2 times 5 L 10 mM sodiumphosphate, pH 7.1, and then stored in the freezer.

For the temperature stability tests, the frozen dialysate was thawed and2×2.5 ml samples buffer exchanged into 3.5 ml phosphate-buffered saline(PBS) or 10 mM Tris buffer, pH 8.0 using mini Sephadex G-25 gelfiltration columns. Buffer exchange into the 10 mM Tris buffer resultedin a precipitate (which contained some activity) which was removed bycentrifugation. Samples of each preparation were then placed in a 60° C.hot block, a 40° C. water bath or a 25° C. incubator. Samples were takenat 1, 2 and 4.3 hours and analysed for residual lactamase activity. Thefollowing results were obtained after 4.3 hours incubation:

    ______________________________________                                                      Temperature                                                                             Residual Activity                                     Buffer        (° C.)                                                                           (% of start)                                          ______________________________________                                        PBS           25        97                                                    PBS           40        87                                                    PBS           60        32                                                    Tris (pH 8)   25        110                                                   Tris (pH 8)   40        105                                                   Tris (pH 8)   60        45                                                    ______________________________________                                    

By comparison, the Pseudomonas fluorescens γ-lactamase described byBrabban et al, supra, lost up to 70-80% of its activity over 4 hours at37° C. The novel enzyme is clearly much more temperature-stable. Thisopens up the possibility of immobilising the enzyme onto a solid supportand re-using it in many biotransformations, thereby greatly reducing itscost impact on the process.

5. Whole-cell Biotransformation

Frozen cell paste (25 g), obtained in a similar fermentation to thatdescribed in Example 3, excepting that the final enzyme yield in thiscase was measured to 0.67 U.ml⁻¹, was thawed and stirred in 50 mM KH₂PO₄ (300 ml, pH7). γ-Lactam (100 g) was added as solid to this, then thereaction stirred at 25° C. for 24 hours. Celite (28 g) thenpolyethylenimine (28 ml of 5% solution in water) were added, followed byisopropanol (175 ml). After stirring for a further 10 minutes, thesolids were removed by filtration, then the filtrate evaporated in vacuoto 200 ml volume. The aqueous was extracted 5 times with dichloromethane(200 ml), then the organic extracts dried using anhydrous MgSO₄. Thefilter-cake was washed with acetone (150 ml) and the extract dried (withanhydrous MgSO₄), then all the combined organic fractions evaporated invacuo to dryness. This yielded 44.3 g of an off-white solid, which wasanalysed to be (-) lactam having an ee of >99%.

This biotransformation could be carried out at a very high substrateconcentration (1 g substrate per 3 ml buffer) and could still providecomplete hydrolysis of the (+) lactam enantiomer. This is thereforehighly volume-efficient, which enables the (-) lactam to be produced ina minimal volume, thus reducing liquid handling requirements andreducing batch biotransformation reactor volume requirements.

6. Identification and Isolation of the Gene

A quantity of cell paste (500 mg) was treated by the addition of TESSbuffer (50 mM Tris.HCl [pH 8.0], 10 mM EDTA, 25 mM NaCl, 25% w/vSucrose) supplemented with lysozyme (1.5 mg ml⁻¹). This treatment wascarried out at 37° C. for 1 hr and the resulting sphereoplasts werelysed by the addition of 10% SDS (1.5% final conc.) To the cell lysate,solid caesium chloride was added at 1 g ml⁻¹. Once dissolved, ethidiumbromide was added at 80 μg ml⁻¹ final conc. The suspension was thenloaded into Sorvall Ultracrimp ultracentrifuge tubes and a gradient wasestablished by centrifugation at 30,000 rpm at 20° C. for 72 hrs. Onceresolved and visualised by an intense ethidium bromide band, the genomicDNA was removed by syringe. Ethidium bromide was removed by extractionwith caesium chloride-saturated butanol. Finally, the genomic DNA wasdialysed in 10,000 volumes of TE buffer (10 mM Tris.HCl, 1 mM EDTA [pH8.0]) with two changes.

A genomic library was prepared by a time-course partial restrictiondigest with Sau3A I (Promega Corp.) restriction endonuclease. Horizontalagarose gel electrophoresis resolved DNA fragments in the range of1.0-4.0 kb. These fragments were excised by electroelution in TBE (16 mMTris. HCl [pH 8.0], 8 mM Boric acid, 400 μM EDTA) at 25 mA current. Theeluted DNA fragments were purified by extraction with an equal volume ofTris-buffered phenol:chloroform and ethanol precipitation. The Sau3A Ipartial genomic DNA fragments were ligated into pUC19; see Yanish-Peronet al, Gene 33:103-119 (1985). The cloning vector pUC19 had beenpreviously linearised by BamHI(Promega Corp.) restriction digestion and5'-phosphate groups were removed by Calf Intestinal Alkaline Phosphatase(Promega Corp.) to prevent re-ligation. Ligations were carried out at14° C. with various ratios of vector and genomic fragments using T4 DNAligase (Boehringer Mannheim Ltd). Ligation reactions were transformedinto Max Efficiency E. coli DH5α (Gibco BRL Life Sciences), transformedE. coli were plated onto Tryptone Soya Agar (Oxoid Ltd) supplementedwith ampicillin (100 μg ml⁻¹), X-Gal (50 μg ml⁻¹), and 1 mM IPTG. Afterovernight incubation at 37° C., transformed E. coli colonies wereadsorbed onto Whatman 2 filter paper discs impregnated with 20 mg ml⁻¹(+)-lactam in methanol. Filters were incubated at room temperature for 4hrs and developed with 2% w/v ninhydrin in acetone. After developing at60° C., a distinctive brown halo upon a purple background, indicative ofamino acid production, could be clearly seen around a single colony. Thesingle lactamase-expressing clone was isolated and lactamase activitywas verified by Achiral and Chiral HPLC assay.

7. Characterisation and Sequencing of Lactamase Gene

Plasmid DNA was prepared from the lactamase-expressing clone.Restriction digest analysis showed the presence of a 1.9 kb Sau3A Irestriction fragment. DNA sequence analysis of the inserted fragmentshowed this fragment to incorporate a open reading frame (ORF) of 1.6 kbwhich, when driven by the upstream lac promoter of pUC19, translates toa protein of 575 residues (61 kDa.); see the Sequence Listing. Thededuced amino acid sequence of the translated ORF shows >65% homology tothe acetamidase from Mycobacterium smegmatis and Methylophilusmethylotrophus. These enzymes have been shown to hydrolyse short chainfatty acylamides; see Draper, J. Gen. Microbiol. 46:111-123 (1969).

With reference to the Sequence Listing, the 1.9 kb lactamase fragmentresides within the two preserved BamHI restriction sites. Sequence 5' tothe insert incorporates the lac promoter and ribosome-binding site ofpUC19.

The pUC19 construct carrying the lactamase gene was subsequentlymodified by the insertion of the cer element from the wild-type E.coliplasmid ColE 1. This construct was designated pPET1.

As will be understood, E. coli plasmid pPET1 was derived from pUC19,which harbours a 1.9 kb Sau3A I genomic fragment from Comomonasacidovorans ligated into the BamHI restriction site. The cer stabilityelement of the wild type plasmid ColE 1 was inserted 3' to the lactamasefragment via BamHI (partial) and NdeI restriction.

8. Growth of Recombinant Lactamase

Recombinant E. coli strain was inoculated into a 1 litre baffled shakeflask containing 100 ml TSB medium (Oxoid Ltd.) supplemented withampicillin (100 μg ml⁻¹). The flask and inoculum were incubated for 16hr at 37° C., shaking at 300 rpm in orbital shaker (25 mm throw). Theseed culture was inoculated (1%) into a 2.8 liter laboratory bioreactorvessel containing 1.5 liters TSB medium. The temperature was maintainedat 25° C., pH 7.0, and dissolved O₂ tension at >50%. Growth wasmonitored at 520_(nm) optical density against a TSB medium blank. After24 hr growth, cells were harvested by centrifugation (5000 g at 4° C.for 10 min.). Cells were stored at -20° C. until required.

9. Use of Recombinant Cells

The E. coli strain harbouring the recombinant plasmid pPET1, was grownand stored as described above. Cells were resuspended at 10% w/v in 100mM Tris.HCl, pH 7.5. Racemic lactam was resuspended of 100 mg.ml⁻¹ in100 mM Tris.HCl, pH 7.5. Reaction conditions for the biotransformationof (+)-lactam were 10 mg ml⁻¹ of racemic lactam mixed with 0.1% w/vrecombinant cells in 100 mM Tris.HCl, pH 7.5. The suspension was reactedat 25° C., shaking at 225 rpm for 1 hr. HPLC analysis after 1 hrreaction showed the conversion of 30% of (+)-lactam to acid with aselectivity of >95% ee.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 2                                             - (2) INFORMATION FOR SEQ ID NO: 1:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1951 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                   #acidovorans) ORGANISM: Comamonas                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION:49..1773                                               #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #ACC ATG      57GGATGTG AGCGATACAA TTTCACACAG GAACAGCT ATG                    #                 Met - # Thr Met                                             # 1                                                                           - ATA ACG CCA AGC TTG CAT GCC TCG GCA GGT CG - #G ACT CTA GAG GAT CCG          105                                                                          Ile Thr Pro Ser Leu His Ala Ser Ala Gly Ar - #g Thr Leu Glu Asp Pro           #      15                                                                     - TTT TTT CCC ACT GCC ATC GCA AGG AGC ACA CC - #A TGG CCG GAA ACC CTG          153                                                                          Phe Phe Pro Thr Ala Ile Ala Arg Ser Thr Pr - #o Trp Pro Glu Thr Leu           # 35                                                                          - ATC AAG GTC GAT CTC AAC CAG TCC CCC TAC GA - #C AAC CCG CAG GTG CAC          201                                                                          Ile Lys Val Asp Leu Asn Gln Ser Pro Tyr As - #p Asn Pro Gln Val His           #                 50                                                          - AAC CGC TGG CAT CCC GAC ATT CCC ATG GCG GT - #C TGG GTG GAG CCG GGC          249                                                                          Asn Arg Trp His Pro Asp Ile Pro Met Ala Va - #l Trp Val Glu Pro Gly           #             65                                                              - GCG GAG TTC AAG CTG GAG ACC TAT GAC TGG AC - #C GGC GGC GCC ATC AAG          297                                                                          Ala Glu Phe Lys Leu Glu Thr Tyr Asp Trp Th - #r Gly Gly Ala Ile Lys           #         80                                                                  - AAC GAC GAC AGC GCC GAA GAC GTG CGC GAC GT - #G GAT CTG TCC ACC GTC          345                                                                          Asn Asp Asp Ser Ala Glu Asp Val Arg Asp Va - #l Asp Leu Ser Thr Val           #     95                                                                      - CAC TTC CTG TCC GGC CCC GTG GGC GTG AAG GG - #C GCG CAG CCC GGC GAC          393                                                                          His Phe Leu Ser Gly Pro Val Gly Val Lys Gl - #y Ala Gln Pro Gly Asp           100                 1 - #05                 1 - #10                 1 -       #15                                                                           - CTG CTG GTG GTG GAC CTG CTG GAC ATC GGC GC - #G CGC GAC GAC AGC CTC          441                                                                          Leu Leu Val Val Asp Leu Leu Asp Ile Gly Al - #a Arg Asp Asp Ser Leu           #               130                                                           - TGG GGC TTC AAC GGC TTT TTC TCC AAG CAG AA - #T GGC GGC GGC TTC CTG          489                                                                          Trp Gly Phe Asn Gly Phe Phe Ser Lys Gln As - #n Gly Gly Gly Phe Leu           #           145                                                               - Asp Glu His Phe Pro Leu Ala Gln Lys Ser Il - #e Trp Asp Phe His Gly         #       160                                                                   - ATG TTC ACC AAG AGC CGC CAC ATC CCC GGC GT - #C AAC TTC GCA GGC CTC          585                                                                          Met Phe Thr Lys Ser Arg His Ile Pro Gly Va - #l Asn Phe Ala Gly Leu           #   175                                                                       - ATC CAC CCG GGC CTG ATC GGC TGC CTG CCC GA - #C CCC AAG ATG CTG GCC          633                                                                          Ile His Pro Gly Leu Ile Gly Cys Leu Pro As - #p Pro Lys Met Leu Ala           180                 1 - #85                 1 - #90                 1 -       #95                                                                           - AGC TGG AAT GAG CGC GAG ACC GGC CTC ATC GC - #C ACC GAC CCC GAC CGC          681                                                                          Ser Trp Asn Glu Arg Glu Thr Gly Leu Ile Al - #a Thr Asp Pro Asp Arg           #               210                                                           - ATT CCC GGC CTG GCC AAC CCG CCC AAC GCC AC - #C ACC GCC CAC ATG GGC          729                                                                          Ile Pro Gly Leu Ala Asn Pro Pro Asn Ala Th - #r Thr Ala His Met Gly           #           225                                                               - CAG ATG CAG GGC GAG GCG CGC GAC AAG GCC GC - #C GCC GAA GGC GCA CGC          777                                                                          Gln Met Gln Gly Glu Ala Arg Asp Lys Ala Al - #a Ala Glu Gly Ala Arg           #       240                                                                   - ACC GTG CCG CCG CGC GAG CAC GGC GGC AAC TG - #C GAC ATC AAG GAC CTC          825                                                                          Thr Val Pro Pro Arg Glu His Gly Gly Asn Cy - #s Asp Ile Lys Asp Leu           #   255                                                                       - TCG CGC GGC TCG CGC GTG TTC TTC CCC GTC TA - #C GTG GAC GGC GCG GGC          873                                                                          Ser Arg Gly Ser Arg Val Phe Phe Pro Val Ty - #r Val Asp Gly Ala Gly           260                 2 - #65                 2 - #70                 2 -       #75                                                                           - CTG AGC GTG GGC GAC CTG CAC TTC AGC CAG GG - #T GAT GGC GAG ATC ACC          921                                                                          Leu Ser Val Gly Asp Leu His Phe Ser Gln Gl - #y Asp Gly Glu Ile Thr           #               290                                                           - TTC TGG GGG CCC ATC GAG ATG CCC GGC TGG GT - #G CAC ATG AAG GTC TCG          969                                                                          Phe Trp Gly Pro Ile Glu Met Pro Gly Trp Va - #l His Met Lys Val Ser           #           305                                                               - CTG ATC AAG GGC GGC ATG GCC AAG TAC GGC AT - #C AAG AAC CCC ATC TTC         1017                                                                          Leu Ile Lys Gly Gly Met Ala Lys Tyr Gly Il - #e Lys Asn Pro Ile Phe           #       320                                                                   - AAG CCC AGC CCC ATG ACG CCC AAC TAC CAA GG - #A CTA CCT GAT CTT CGA         1065                                                                          Lys Pro Ser Pro Met Thr Pro Asn Tyr Gln Gl - #y Leu Pro Asp Leu Arg           #   335                                                                       - AGG CAT CTC GGT GGA CGA AAA GGG CAA GCA GC - #A CTA CCT GGA CGT GAC         1113                                                                          Arg His Leu Gly Gly Arg Lys Gly Gln Ala Al - #a Leu Pro Gly Arg Asp           340                 3 - #45                 3 - #50                 3 -       #55                                                                           - CGT GGC CTA CCG CCA GGC CTG CCT GAA CGC CA - #T CGA GTA CCT GAA GAA         1161                                                                          Arg Gly Leu Pro Pro Gly Leu Pro Glu Arg Hi - #s Arg Val Pro Glu Glu           #               370                                                           - ATT CGG CTA CAG CGG CGC CCA GGC CTA CTC GC - #T GCT GGG CAC GGC GCC         1209                                                                          Ile Arg Leu Gln Arg Arg Pro Gly Leu Leu Al - #a Ala Gly His Gly Ala           #           385                                                               - CGT GCA GGG CCA CAT CAG CGG CGT GGT GGA CG - #T GCC CAA TGC CTG CGC         1257                                                                          Arg Ala Gly Pro His Gln Arg Arg Gly Gly Ar - #g Ala Gln Cys Leu Arg           #       400                                                                   - CAC GCT GTG GCT GCC CAC GGA GAT CTT CGA CT - #T CGA CAT CAA TCC CAC         1305                                                                          His Ala Val Ala Ala His Gly Asp Leu Arg Le - #u Arg His Gln Ser His           #   415                                                                       - GGC CGA GGG ACC ACA GAA GAT CAT CAC GGG CG - #G GGT GGA TCT GCC CAT         1353                                                                          Gly Arg Gly Thr Thr Glu Asp His His Gly Ar - #g Gly Gly Ser Ala His           420                 4 - #25                 4 - #30                 4 -       #35                                                                           - CGC CCA GGA CAA GTA AGC CCG GCA TAC GAC AC - #C CGC CAT CCA CCA TTC         1401                                                                          Arg Pro Gly Gln Val Ser Pro Ala Tyr Asp Th - #r Arg His Pro Pro Phe           #               450                                                           - GCC AGA GGC CGC CCA TGC CCA CCT ATG ACT AC - #C ACT GCA CCG CAT GCG         1449                                                                          Ala Arg Gly Arg Pro Cys Pro Pro Met Thr Th - #r Thr Ala Pro His Ala           #           465                                                               - GCG GCT TCG ACG CGC TGC GCA GCC TCT CGC AG - #C GCA ACG AGC CCG CGC         1497                                                                          Ala Ala Ser Thr Arg Cys Ala Ala Ser Arg Se - #r Ala Thr Ser Pro Arg           #       480                                                                   - CCT GCC CCA GCT GCG AGG CGG CCT CGC CCC GC - #G TCT TCG TCA GCG CGC         1545                                                                          Pro Ala Pro Ala Ala Arg Arg Pro Arg Pro Al - #a Ser Ser Ser Ala Arg           #   495                                                                       - CGC GCC TGG CCT GCA CCA GCC CCG AAC AGC GC - #C GCG CCC ACG ACA CCA         1593                                                                          - Arg Ala Trp Pro Ala Pro Ala Pro Asn Ser Al - #a Ala Pro Thr Thr Pro         500                 5 - #05                 5 - #10                 5 -       #15                                                                           - ACG AGC GCG CCC GGC ACG AGC CCA GGC GCT CA - #C GCG ATG TGG CCG AGG         1641                                                                          Thr Ser Ala Pro Gly Thr Ser Pro Gly Ala Hi - #s Ala Met Trp Pro Arg           #               530                                                           - GCA GCT ACG CGC GCA TGC GCC ACC CCA TCG GG - #C TGC GGC TGC TGC AGC         1689                                                                          Ala Ala Thr Arg Ala Cys Ala Thr Pro Ser Gl - #y Cys Gly Cys Cys Ser           #           545                                                               - GGC GCC AGC AAG CGC GGC TCC ACG GTC ACG GC - #G CCC AAC GGC GCC AAG         1737                                                                          Gly Ala Ser Lys Arg Gly Ser Thr Val Thr Al - #a Pro Asn Gly Ala Lys           #       560                                                                   - ACC TTC CCG ACC AAG CGG CCC TGG ATG ATC AG - #C CAC TGA CCG CGG A           1783                                                                          Thr Phe Pro Thr Lys Arg Pro Trp Met Ile Se - #r His                           #   575                                                                       - CCCTGCGCCG CACCAATGAC AAGGGCCCGC GACGCGGGCC TTTGTCCTGC CT - #GGCCGTAC       1843                                                                          - CGCTCAGTGC ACGGCGCCGA TGAAGCCGGC CAGCTCCGGC GTCTGCGGGT TG - #GCGAACAG       1903                                                                          #              1951CGCT TTCGTGGATC CCCGGTACCG AATCGATC                        - (2) INFORMATION FOR SEQ ID NO: 2:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 575 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Met Thr Met Ile Thr Pro Ser Leu His Ala Se - #r Ala Gly Arg Thr Leu         #                 15                                                          - Glu Asp Pro Phe Phe Pro Thr Ala Ile Ala Ar - #g Ser Thr Pro Trp Pro         #             30                                                              - Glu Thr Leu Ile Lys Val Asp Leu Asn Gln Se - #r Pro Tyr Asp Asn Pro         #         45                                                                  - Gln Val His Asn Arg Trp His Pro Asp Ile Pr - #o Met Ala Val Trp Val         #     60                                                                      - Glu Pro Gly Ala Glu Phe Lys Leu Glu Thr Ty - #r Asp Trp Thr Gly Gly         # 80                                                                          - Ala Ile Lys Asn Asp Asp Ser Ala Glu Asp Va - #l Arg Asp Val Asp Leu         #                 95                                                          - Ser Thr Val His Phe Leu Ser Gly Pro Val Gl - #y Val Lys Gly Ala Gln         #           110                                                               - Pro Gly Asp Leu Leu Val Val Asp Leu Leu As - #p Ile Gly Ala Arg Asp         #       125                                                                   - Asp Ser Leu Trp Gly Phe Asn Gly Phe Phe Se - #r Lys Gln Asn Gly Gly         #   140                                                                       - Gly Phe Leu Asp Glu His Phe Pro Leu Ala Gl - #n Lys Ser Ile Trp Asp         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Phe His Gly Met Phe Thr Lys Ser Arg His Il - #e Pro Gly Val Asn Phe         #               175                                                           - Ala Gly Leu Ile His Pro Gly Leu Ile Gly Cy - #s Leu Pro Asp Pro Lys         #           190                                                               - Met Leu Ala Ser Trp Asn Glu Arg Glu Thr Gl - #y Leu Ile Ala Thr Asp         #       205                                                                   - Pro Asp Arg Ile Pro Gly Leu Ala Asn Pro Pr - #o Asn Ala Thr Thr Ala         #   220                                                                       - His Met Gly Gln Met Gln Gly Glu Ala Arg As - #p Lys Ala Ala Ala Glu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Gly Ala Arg Thr Val Pro Pro Arg Glu His Gl - #y Gly Asn Cys Asp Ile         #               255                                                           - Lys Asp Leu Ser Arg Gly Ser Arg Val Phe Ph - #e Pro Val Tyr Val Asp         #           270                                                               - Gly Ala Gly Leu Ser Val Gly Asp Leu His Ph - #e Ser Gln Gly Asp Gly         #       285                                                                   - Glu Ile Thr Phe Trp Gly Pro Ile Glu Met Pr - #o Gly Trp Val His Met         #   300                                                                       - Lys Val Ser Leu Ile Lys Gly Gly Met Ala Ly - #s Tyr Gly Ile Lys Asn         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Pro Ile Phe Lys Pro Ser Pro Met Thr Pro As - #n Tyr Gln Gly Leu Pro         #               335                                                           - Asp Leu Arg Arg His Leu Gly Gly Arg Lys Gl - #y Gln Ala Ala Leu Pro         #           350                                                               - Gly Arg Asp Arg Gly Leu Pro Pro Gly Leu Pr - #o Glu Arg His Arg Val         #       365                                                                   - Pro Glu Glu Ile Arg Leu Gln Arg Arg Pro Gl - #y Leu Leu Ala Ala Gly         #   380                                                                       - His Gly Ala Arg Ala Gly Pro His Gln Arg Ar - #g Gly Gly Arg Ala Gln         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Cys Leu Arg His Ala Val Ala Ala His Gly As - #p Leu Arg Leu Arg His         #               415                                                           - Gln Ser His Gly Arg Gly Thr Thr Glu Asp Hi - #s His Gly Arg Gly Gly         #           430                                                               - Ser Ala His Arg Pro Gly Gln Val Ser Pro Al - #a Tyr Asp Thr Arg His         #       445                                                                   - Pro Pro Phe Ala Arg Gly Arg Pro Cys Pro Pr - #o Met Thr Thr Thr Ala         #   460                                                                       - Pro His Ala Ala Ala Ser Thr Arg Cys Ala Al - #a Ser Arg Ser Ala Thr         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Ser Pro Arg Pro Ala Pro Ala Ala Arg Arg Pr - #o Arg Pro Ala Ser Ser         #               495                                                           - Ser Ala Arg Arg Ala Trp Pro Ala Pro Ala Pr - #o Asn Ser Ala Ala Pro         #           510                                                               - Thr Thr Pro Thr Ser Ala Pro Gly Thr Ser Pr - #o Gly Ala His Ala Met         #       525                                                                   - Trp Pro Arg Ala Ala Thr Arg Ala Cys Ala Th - #r Pro Ser Gly Cys Gly         #   540                                                                       - Cys Cys Ser Gly Ala Ser Lys Arg Gly Ser Th - #r Val Thr Ala Pro Asn         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Gly Ala Lys Thr Phe Pro Thr Lys Arg Pro Tr - #p Met Ile Ser His             #               575                                                           __________________________________________________________________________

We claim:
 1. A method for the stereoselective hydrolysis of a mixture of enantiomers of 2-azabicyclo[2.2.1]hept-5-en-3-one, which comprises contacting the mixture with an enzyme capable of hydrolysing an enantiomer of the bicyclic lactam, 2-azabicyclo[2.2.1]hept-5-en-3-one, the enzyme having one or more of the following properties:greater than 85% retention of activity after being held at 40°C. for 4 hours or greater than 30% activity after being held at 60° C. for 4 hours; hydrolysis at an initial concentration of 100 g racemic lactam plus 300 ml buffer and continuing to at least 90% hydrolysis of the (+) lactam with less than 5% hydrolysis of the (-) lactam; wherein the enzyme is prepared by culturing a microorganism capable of expressing an enzyme comprising the amino acid sequence shown in SEQ ID NO:2, or a fragment thereof, capable of hydrolysing an enantiomer of the bicyclic lactam, 2-azabicyclo[2.2.1]hept-5-en-3-one.
 2. The method, according to claim 1, which additionally comprises separating residual (-) lactam from the (+) amino acid formed by hydrolysis.
 3. The process according to claim 2, which additionally comprises converting the (-) lactam to a carbocyclic nucleoside. 